Numerous methods and systems have been developed for the detection and quantitation of analytes of interest in biochemical and biological substances. Methods and systems which are capable of measuring trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins are of great value to researchers and clinicians.
A very substantial body of art has been developed based upon the well known binding reactions, e.g., antigen-antibody reactions, nucleic acid hybridization techniques, and protein-ligand systems. The high degree of specificity in many biochemical and biological binding systems has led to many assay methods and systems of value in research and diagnostics. Typically, the existence of an analyte of interest is indicated by the presence or absence of an observable “label” attached to one or more of the binding materials.
Chemiluminescent assay techniques where a sample containing an analyte of interest is mixed with a reactant labeled with a chemiluminescent label have been developed. The reactive mixture is incubated and some portion of the labeled reactant binds to the analyte. After incubation, the bound and unbound fractions of the mixture are separated and the concentration of the label in either or both fractions can be determined by chemiluminescent techniques. The level of chemiluminescence determined in one or both fractions indicates the amount of analyte of interest in the biological sample.
Electrochemiluminescent (ECL) assay techniques are an improvement on chemiluminescent techniques. They provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. In such techniques, the incubated sample is exposed to a voltammetric working electrode in order to trigger luminescence. In the proper chemical environment, such electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to U.S. application Serial No. 06/789,113, filed October 24, 1985 now U.S. Patent No. 5,238,808, pending application Serial No. 07/993,530 filed Dec. 17, 1992, which is a continuation of Serial No. 07/675,019 filed Mar. 25, 1991, now abandoned, which is a continuation of application Serial No. 06/858,354 filed Apr. 30, 1986, now abandoned, PCT published application number U.S. 87/00987 filed Apr. 30, 1987, now abandoned, which is a continuation-in-part of U.S. national phase PCT application Ser. No. 07/369,560 filed Dec. 18, 1987 now abandoned in favor of pending continuation application Ser. No. 08/195,825 filed Feb. 10, 1994, and U.S. application Ser. No. 07/117,017 filed Nov. 4, 1987, now abandoned, in favor of U.S. application Serial No. 07/539,389 filed Jun. 18, 1990, which is a continuation of application Ser. No. 07/266,882, filed November 3, 1988, now abandoned, which is a continuation-in-part of application Serial No. 06/858,354 filed Apr. 30, 1986, now abandoned. Reference is also made to PCT published application US85/02153 (WO86/02734), the content of which is incorporated by reference. The disclosure of the aforesaid applications is incorporated by reference.
It would be desirable to carry out electrochemiluminescent assays without the need for a separation step during the assay procedure and to maximize the signal modulation at different concentrations of analyte so that precise and sensitive measurements can be made. Among prior art methods for nonseparation assays are those which employ microparticulate matter suspended in the assay sample to bind one or more of the binding components of the assay. U.S. Pat. No. 4,305,925 relates to the detection and determination of clinically relevant proteins and peptides by means of nephelometric and turbidometric methods. The methods disclosed involve binding the antigen or antibody to latex particles which perform the function of light scattering or adsorption.
U.S. Pat. No. 4,480,042 relates to techniques employing particle reagents consisting of shell-core particles. The shell contains functional groups to which compounds of biological interest can be covalently bonded, and the high refractive index of the core results in high sensitivity to light scattering measurements. The technique is based upon agglutination reactions which result from the reaction of bivalent antibodies with multivalent antigens of interest to produce aggregates which can be detected and/or measured in various ways.
U.S. Pat. No. 4,419,453 likewise relates to the use of colored latex agglutination test methods useful for detecting the presence of immunochemicals such as antibodies and immunogens.
The assay techniques of the prior art and the use of microparticulate matter in the assay medium would not appear applicable for assays wherein a luminescent phenomenon is measured. One would expect that the luminescence from free chemiluminescent or electrochemiluminescent moieties would be absorbed, scattered, or otherwise suffer interference from the microparticulate matter.